1. Field of the Invention
The invention disclosed relates to a multi-purpose laboratory apparatus, in particular a single, multi-purpose, apparatus in which electrophoresis, horizontal and vertical electroblotting and/or electroelution, vacuum blotting, capillary blotting, UV observation of ethidium bromide labeled specimens, UV photography and UV cross linking may each be performed. Additional feature includes buffer circulation, light opaque enclosure, DNA band compaction and DNA elution without cutting DNA band.
2. Description of the Related Art
Gel electrophoresis is a key analytical tool in molecular biology and clinical sciences. The electrophoresis of DNA and other organic molecules accomplished under the influence of an electric field is known. In order to perform this process, a semisolid gel of agarose, polyacrylamide or other similar castable, transparent material, is cast in a mold to produce a thin rectangular slab. Wells, or depressions, are formed in the upper surface of the slab along a line positioned at one end or in the center of the gel slab. The entire gel slab is then placed in a chamber having electrodes at each end. The chamber is then filled with a buffer solution to a level higher than the gel. Selected wells are filled with a solution containing the sample being analyzed, and a high gradient electric field is applied to the gel through the buffer solution. The molecules comprising the samples are caused to separate into sample bands by migrating through the gel slab towards the oppositely charged electrode at different rates depending upon their mobility, a function of molecular charge density and molecular size.
Gels are fragile. Manipulation can often cause a gel to rip or distort, possibly resulting in lost or unreliable experimental results. For example, the sample is typically stained with an intercalating fluorescent reagent e.g. ethidium bromide, which gives the DNA band sharp fluorescence under UV irradiation. An operator using currently known electrophoresis equipment cannot observe ethidium bromide stained DNA sample band migration during electrophoresis or electroblotting. Traditionally, the operator must terminate electrophoresis and transport the gel to a sample evaluation transilluminator utilizing a UV light source to irradiate ethidium bromide stained DNA for visual inspection of sample separation. If additional electrophoresis is required to complete the test, the gel must be returned to the chamber and the process resumed. Subsequently, to obtain a permanent record of the sample separation results, the DNA gel must again be physically transported from the electrophoretic device to a UV transilluminating platform above which the operator mounts a UV sensitive camera for photographing the gel. An inexperienced operator often wastes a number of film exposures during the process of aligning a gel slab on a UV platform before obtaining a good photograph. As stated above, these frequent manipulations can compromise gel integrity and lead to lost or unreliable research data. In addition, when the gel is reinserted into the electrophoresis chamber after observation, a change from the original gel position, or from the original buffer pH, temperature or composition may complete the electrophoretic run under different operating conditions, and possibly lead to further unreliable research data.
The usefulness of electrophoresis depends on producing thin, well defined molecular sample bands. This definition depends, in part, on the manner in which the molecular sample migrates from the sample well along the gel. The sample well, the distribution of sample molecular material in the well, and the electric field all influence band definition. A molecular sample once deposited in a well migrates horizontally through the gel as a vertically oriented rectangle. The sides of the sample rectangle tend to migrate slower than the central portion, resulting in a U-shaped dispersed band. However, a macromolecular sample deposited in the front corner of the well migrates horizontally as a compact line through a comparatively small volume of gel, thus resulting in narrow, straight, well defined bands. Traditionally, an operator must use extreme care in loading of the sample to attempt to obtain a well defined band. However, despite such care, electrophoretic results are often diminished by the presence of U-shaped dispersed macromolecular sample bands.
Subsequent to electrophoresis, it is common to transfer the molecular sample bands from the gel to a suitable membrane for further purification, processing or analysis. This transfer process is generally known as electroblotting. A vertically or horizontally oriented high gradient electric field is applied to the gel to accomplish transfer of sample bands to an adjacent membrane. Particular transfer techniques, e.g. northern and southern blotting, typically employ nitrocellulose or nylon membranes. The adhered sample may then be hybridized to a probe for further analysis or processing. Electroelution, is a technique which typically employs Di-Ethyl Amino Ethyl Cellulose (DEAE cellulose) paper membrane. DEAE cellulose paper adsorbs sample bands which may then be recovered from the DEAE cellulose paper for further processing like washing and eluting with various solutions. Vacuum blotting is another method for transfer of a molecular sample from the gel to an adjacent membrane. Vacuum blotting is accomplished by exposing the gel to a mild vacuum to cause transfer of molecular sample from the gel to an adjacent membrane. Existing electrophoresis units are not equipped to handle vacuum blotting. Therefore, the gel must be transported, according to current methodology, from the electrophoresis unit to another device.
Capillary blotting is similar to the electro-blotting methods described above, except that transfer of DNA from the gel to an adjacent membrane is accomplished by capillary movement of a buffer in a direction from the gel towards the adjacent membrane, causing the transfer of DNA from the gel to an adjacent membrane. Existing electrophoresis units are not equipped to handle capillary blotting processes. Therefore, the gel must be transported, according to current methodology, from the electrophoresis unit to another device.
UV cross linking of DNA molecules is a process whereby DNA molecules which have been transferred to a nitrocellulose or nylon membrane are made to chemically bond to the nitrocellulose or nylon molecules in the membrane by exposure to UV radiation. Traditionally, for this bonding process, another separate piece of laboratory apparatus must be used.
All the above-mentioned electroblotting techniques require that the gel be removed from the electrophoresis device and deposited in a denaturation and neutralization bath before transfer. After bathing, the gel is deposited upon an electro-blot membrane. The membrane and gel are then sandwiched between absorbent layers and transferred to a separate electro-blot device for electro-transfer.
One device, taught by Shuette in U.S. Pat. No. 5,013,1820, consolidates electrophoresis and electroblotting. However, the gel must still be removed from the Shuette device after electrophoresis for denaturation and assembly of the sandwich. The sandwich is then reinserted into the Shuette device before electro-blotting. Although the Shuette device consolidates electrophoresis and electroblotting procedures, the number of manipulation steps necessary to accomplish electro-transfer and which are required to take place outside the apparatus remain the same as in separate apparatus.
A second device, disclosed in U.S. Pat. No. 4,657,655 to Smoot et al., utilizes a gel-casting tray which may be mounted either in an electrophoresis chamber for separation processes or in a transilluminator for visualization and photography. The tray has vertically slidable side gates to go from a casting position to a lower position to locate the tray in each respective unit.
No single work station exists in which essentially all the above mentioned experimental procedures, can be performed. Laboratories must purchase various separate units, thus increasing cost and space requirements. Traditional techniques involving gel transfer also repeatedly expose operators to a known carcinogen, ethidium bromide.
Therefore, it is an object of the present invention to provide an apparatus which consolidates single or multiple gel electrophoresis, whole gel or single band electroeluting, electroblotting, capillary blotting, vacuum blotting, UV cross linking, UV observation, mini dark room and UV photography into one multipurpose work station.
Another object of the invention is to reduce handling and movement of the gel, reduce operator exposure to ethidium bromide and improve test result reliability.
Another object of the invention is to provide an apparatus to carry out horizontal as well as vertical movement of sample molecules without touching the gel.
It is a further object of the present invention to provide a universal work station apparatus that enables a molecular sample to be compressed to the front lower corner of each formed well for improved definition of molecular sample bands.
Another object of the invention is to provide a design in which the buffer solution is circulated.
Still another object of the present invention is to provide an apparatus for observation of molecular movement for complete electroblotting or electroelution which can be performed safely during UV operation.
Further objectives of the invention will become apparent from the description that follows.